Genetic mutations linked to field‐evolved Cry1Fa-resistance in the European corn borer, Ostrinia nubilalis

Transgenic corn, Zea mays (L.), expressing insecticidal toxins such as Cry1Fa, from Bacillus thuringiensis (Bt corn) targeting Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) resulted in over 20 years of management success. The first case of practical field-evolved resistance by O. nubilalis to a Bt corn toxin, Cry1Fa, was discovered in Nova Scotia, Canada, in 2018. Laboratory-derived Cry1Fa-resistance by O. nubilalis was linked to a genome region encoding the ATP Binding Cassette subfamily C2 (ABCC2) gene; however, the involvement of ABCC2 and specific mutations in the gene leading to resistance remain unknown. Using a classical candidate gene approach, we report on O. nubilalis ABCC2 gene mutations linked to laboratory-derived and field-evolved Cry1Fa-resistance. Using these mutations, a DNA-based genotyping assay was developed to test for the presence of the Cry1Fa-resistance alleles in O. nubilalis strains collected in Canada. Screening data provide strong evidence that field-evolved Cry1Fa-resistance in O. nubilalis maps to the ABCC2 gene and demonstrates the utility of this assay for detecting the Cry1Fa resistance allele in O. nubilalis. This study is the first to describe mutations linked to Bt resistance in O. nubilalis and provides a DNA-based detection method that can be used for monitoring.


Results
Toxin-overlay diet bioassays. The susceptibility of four O. nubilalis strains to Cry1Fa was determined using a toxin-overlay diet bioassay with a non-treated control (0 ng Cry1Fa cm −2 ) and a diagnostic concentration of 200 ng Cry1Fa cm −2 . Mortality of ON-S1 and ON-S2 was 100% with the diagnostic concentration, which was significantly greater than the mortality of NS-R and QC-R (< 25%) and the mortality of all strains in the non-treated control (< 15%) ( Table 1). There was no difference in mortality between concentrations for NS-R and QC-R (Table 1). Based on these results, ON-S1 and ON-S2 were deemed susceptible to Cry1Fa, while NS-R and QC-R were deemed resistant.

Introgression experiment. An introgression experiment was conducted to establish a back-cross strain
that is an isoline of ON-S1 but resistant to Cry1Fa. To do this, ON-S1 was crossed with NS-R1 and the heterozygote F 1 where then interbred. Following this, F 2 , selected for their resistance to Cry1Fa, were back-crossed with ON-S1. This process was repeated 5 times, as shown in Table 2 and described in materials and methods section. The diagnostic concentration of Cry1Fa caused 100% mortality among the offspring of ON-S1 crossings (ON-S1 × F n ), but mortality among the interbred crossing (F n × F n ) was significantly lower, ranging between 70-76% (Table 2). Mortality in all back-crossed strains was significantly greater when exposed to the Cry1Fa diagnostic concentration relative to the non-treated control ( Table 2). These results demonstrate that the Cry1Fa resistance allele is recessive and inheritance followed Mendelian expectations for a single gene ( Table 2). BC-R was established by interbreeding F 10 individuals that survived exposure to the Cry1Fa diagnostic concentration. Exposing this strain to the diagnostic concentration of Cry1Fa caused 17% mortality which did not differ from the non-treated control ( Table 2).
Sequencing. The ABCC2 gene of O. nubilalis contained 24 predicted exons, totaling 4,089 bp of coding sequence. We sequenced ~ 4.0 Kb (> 98%) of the ABCC2 coding region from ON-S1 and NS-R. In total, we found 20 SNPs (~ 1 per 200 bp) between resistant and susceptible individuals and relative to the reference gene. Most SNPs were synonymous substitutions that did not alter the amino acid sequence, and a few were fixed between susceptible and resistant strains. Presented in Table 3 are six nonsynonymous SNPs, one of which is a two nucleotide base deletion (SNP5), fixed between resistant and susceptible strains and the predicted amino acid change. The predicted location of these SNPs on the ABCC2 protein are presented in Fig. 1. SNP4 resulted in an amino acid change from glycine in the susceptible strains to serine in the resistant strains. This change is predicted to be on ECL4 (Fig. 1). The two nucleotide base deletion (SNP5) induced a frameshift and a premature stop codon www.nature.com/scientificreports/ at amino acid 982, truncating the protein and resulting in the loss of the 11th and 12th transmembrane helices and NBD2 (Fig. 1). The sequenced data were compared to the publicly available sequence of the laboratory-derived Cry1Faresistant O. nubilalis described in Vellichirammal et al. 18 where we found 18 of the 20 identified SNPs present between the susceptible and Cry1Fa-resistant strains. Of the nonsynonymous SNPS, all five were fixed between the susceptible and laboratory-derived Cry1Fa-resistant strains (Table 3). However, SNP5 documented in our field-evolved Cry1Fa-resistant strains was not present in the laboratory-derived Cry1Fa-resistant O. nubilalis described in Vellichirammal et al. 18 (Table 3). A non-synonymous SNP downstream of SNP6 was present in the laboratory-derived Cry1Fa-resistant strain described in Vellichirammal et al. 18 (hereinafter referred to as SNPV) that resulted in protein truncation due to a premature stop codon, with the predicted loss of the NBD2 (Table 3).
Genotyping assays. A restriction enzyme digest genotyping assay was developed to detect the six nonsynonymous SNPs found in the sequenced resistant strains. The Cry1Fa-resistant strains were homozygous for the six non-synonymous SNPs, while the non-synonymous SNPs were only detected at a frequency of 0.04 in ON-S1 and 0.00 for ON-S2 (Table 4). BC-R was also homozygous for the six non-synonymous SNPs (Table 4).
Randomly selected individuals from the last back-cross assay were genotyped and digest results are presented in Fig. 2. Restriction enzyme digests of randomly selected individuals from the last back-cross assay showed that individuals who survived exposure to non-treated control were in Hardy-Weinberg equilibrium (χ 2 (2, n = 25) = 0.4400, P = 0.8025), with 24% homozygous for the six non-synonymous SNPs associated with Cry1Fa susceptibility (p 2 ), 52% heterozygous (2pq), and 24% homozygous for the six non-synonymous SNPs (q 2 ) (Fig. 2a). The percentage of genotyped individuals that died after exposure to diagnostic concentration were 56, 40, and 4% for homozygous susceptible (p2), heterozygous (2pq), and homozygous resistant (q 2 ), respectively  Table 3. Allelic differences in the ABCC2 gene and the predicted amino acid changes between Cry1Fasusceptible (Cry1Fa-S) and Cry1Fa-resistant (Cry1Fa-R) Ostrinia nubilalis strains and the presence of the documented allelic differences in the laboratory-derived Cry1Fa-resistant strain. 1 Cry1Fa-R alleles present in either field-evolved, laboratory-derived, or both Cry1Fa-resistant strains. 2 Based on available data obtained from Vellichirammal et al. (2015). 3 SNPV denotes a mutational change present in the laboratory-derived Cry1Fa-resistant strain but not in the field-derived Cry1Fa-resistant strains.  Fig. 2b), which deviated from Hardy-Weinberg equilibrium (χ 2 (2, n = 25) = 6.1200, P = 0.0469). Individuals that survived exposure to diagnostic concentration were 100% homozygous for the six non-synonymous SNPs (q 2 ) (Fig. 2c).

Discussion
Field-evolved Cry1Fa-resistance in O. nubilalis is closely linked to mutations in the ABCC2 gene which functions as a receptor for the Cry1Fa toxin in susceptible insects. This is the first study to describe mutations linked to Bt resistance in O. nubilalis. Analogous comparisons between laboratory-derived and field-evolved Cry1F resistant O. nubilalis show striking similarities at the gene-level and with some of the detected mutations.
Genotyping results using a Cry1Fa-resistant back-crossed strain provide support for linkage of six mutations to field-evolved Cry1Fa-resistance in O. nubilalis. The linkage tests detected no heterozygote survival among the back-crossed larvae exposed to the Cry1Fa toxin, suggesting that the resistance trait is completely recessive.
Survival of approximately 25% of back-crossed individuals exposed to a diagnostic dose of Cry1Fa indicates that the resistance trait is controlled by a single gene. This result is similar to that reported within a laboratoryderived Cry1Fa-resistant O. nubilalis strain 17 .
A single nucleotide substitution that resulted in an amino acid change in ECL4 was present among the fieldderived resistant strains in this study and in the laboratory-derived Cry1Fa-resistant strain 18 . ECL4 has been suggested as a potential binding site for Cry1 toxins as studies have shown that mutations in ECL4 confer resistance to Cry1 toxins in S. frugiperda, S. exigua, Helicoverpa armigera (Hübner), and B. mori [13][14][15][16]24,26,27 . However, the mutation described in this study requires further investigation to better indicate its link to the binding site of Cry1Fa on the ABCC2 protein.
Another mutation described in this study is a two nucleotide base deletion that induces a frameshift, resulting in a premature stop codon, truncating the protein, and resulting in the loss of the last third of the second transmembrane domain including the NBD2. These nucleotide deletions were also present among the BC-R and QC-R strains tested, but not in the laboratory-derived Cry1Fa-resistant strain. However, SNPV was present  Table 4. Allele frequencies of six non-synonymous SNPs associated with field-evolved Cry1Fa-resistance in Cry1Fa-susceptible, Cry1Fa-resistant, and back-crossed strains of Ostrinia nubilalis using digest enzymes. 1 Total number of individuals genotyped. 2 Cut sites are described in Table S3. 3 BC-R represents a back-crossed Cry1Fa-resistant strain that is 96% genetically similar to ON-S1 but contains the Cry1Fa-resistant trait from crossing with NS-R. www.nature.com/scientificreports/ in the laboratory-derived Cry1Fa-resistant strain and also resulted in protein truncation, with the predicted loss of the NBD2. High-level resistance to Cry1Fa was documented using a CRISPR-mediated ABCC2 gene knockout that resulted in the loss of both ECL4 and NBD2 in O. furnacalis and S. exigua 23,28 . Furthermore, mutational changes associated with field-evolved Cry1Fa-resistant S. frugiperda from Puerto Rico and Florida resulted in ABCC2 protein truncation with the loss of the second transmembrane domain, including ECL4 and NBD2 6,7 . Mutations in NBD2, including the NBD2-deletion, did not, on their own, alter Cry1Fa susceptibility in baculovirus-free insect cell expression of ABCC2 variants of S. frugiperda 13 . Using functional genetic testing such as baculovirus-free insect cells expressing the mutations documented in this study, either alone or in combination, can help determine the mutations responsible for Cry1Fa-resistance in O. nubilalis and determine the mechanisms behind Cry1Fa resistance. ABCC2 has been described as a binding receptor for Cry1Ab, Cry1Ac, and Cry1A.105, in addition to Cry1Fa 15,16,29 . Mutations to ABCC2 resulting in Cry1Fa-resistance may also alter O. nubilalis susceptibility to other Cry1 toxins if resistance was due to an alteration of the shared binding sites 30,31 . CRISPR-mediated ABCC2 gene knockout in O. furnacalis resulted in over 300-fold resistance to Cry1Fa and 4-eightfold resistance to Cry1Ab and Cry1Ac 23 . Knocking out ABCC2 in S. frugiperda conferred over 120-fold resistance to Cry1Fa and Cry1Ab 32 . In a cell expression study of ABCC2 mutations documented in S. frugiperda, single nucleotide substitutions in ECL4 conferred over 630-fold resistance to Cry1Fa and over ninefold resistance to Cry1Ab and Cry1A.105 13 . ABCC2 as a shared receptor among Cry1 toxins was also documented in other Lepidopteran species such as B. mori, H. armigera, and S. exigua 15,16,22,24,28 .
The frequency of the Cry1Fa resistance allele before the adoption of Cry1Fa corn in NS is unknown as Bt resistance monitoring was not conducted in the Maritimes region before 2018. The Cry1Fa resistance allele frequency (0.04) observed in the ON-S1 strain in the present study is greater than that reported in the U.S. corn belt (> 10 -3 ) 33 . The Cry1Fa resistance allele frequency in U.S. corn belt was estimated using an F 1 , where field-collected individuals were crossed with a resistant laboratory strain, and F 2 screening approach to estimate , and (c) survived exposure to diagnostic concentration (200 ng Cry1Fa cm −2 ) of the F2BC-RxON-S1 as determined using restriction enzyme digests. Black, grey, and white bars indicate homozygotes for the alleles associated with Cry1Fa susceptibility (p 2 ), heterozygotes (2pq), and homozygotes for the alleles associated with Cry1Fa resistance (q 2 ), respectively. www.nature.com/scientificreports/ Cry1Fa resistance allele frequency. The results revealed the presence of the Cry1Fa resistance allele frequency at higher than estimated levels even during the first year of commercialization of Cry1Fa corn; however, there was no indication of that frequency increasing over the 7 years of the study 33 . The origin of this allele is also unclear. However, B. thuringiensis is ubiquitous in the environment 34 and contact with this bacterium throughout O. nubilalis evolution may have resulted in the presence of alleles conferring resistance to Bt toxins 3 . Evolution of resistance is likely to capitalize on alleles already present in the population 3 . The analogous comparison of the genetic basis of Bt resistance between laboratory-derived and field-evolved Cry1Fa resistance in O. nubilalis show a great deal of similarities at both the gene-level and with some of the detected mutations. To our knowledge, only one other study conducted analogous comparisons between laboratory-derived and field-evolved Bt resistance, finding striking similarities between the two strains of P. gossypiella 9 . These similarities highlight the usefulness of laboratory selection for identifying genes important in field-evolved Bt resistance, but may not necessarily capture the specific mutations in those genes. The present study is the first to describe mutations in O. nubilalis linked to Bt resistance and is one of only three studies to identify molecular genetic basis of practical Bt resistance. Using the genotyping method described in this study, changes in the frequency of Cry1Fa-resistance across the range of O. nubilalis can be detected, allowing for a more rapid implementation of Bt resistance management strategies.  Table S1). Larvae were reared in 36 cm diameter plastic tubs containing meridic diet with corrugated cardboard rings placed above the diet surface as described by Smith et al. 11 . Once the majority of larvae had pupated in the rings, they were transferred to cages (30 cm × 30 cm × 60 cm) with waxed paper sheets placed on top of the mesh roof for oviposition. Waxed sheets were replaced daily and placed into plastic sweater boxes lined with moistened paper towels. All rearing stages and bioassays were maintained at 26:18 °C, 60% RH, and 16:8 (L:D) h photoperiod.

Methods and material
Toxin-overlay diet bioassays. The susceptibility of O. nubilalis strains to Cry1Fa was determined using toxin-overlay diet bioassays. Diet was prepared in the same manner as for rearing 11 . Using a repeater pipette, 1 mL of diet was dispensed into each well of a 128-well bioassay tray (Bio-16, CD International, Pitman, NJ) resulting in a surface area of 2.0 cm 2 . After the diet has solidified, wells were covered with adhesive ventilated lids (Frontier Agricultural Sciences, Newark, DE), and stored at 4 °C. Lyophilized toxin standard containing > 95% purity of activated Cry1Fa, produced from Escherichia coli (Migula) containing the Cry1F gene, was obtained from M. Pusztai-Carey (Case-Western University, Cleveland, OH), and stored at −80 °C. Cry1Fa toxin was constituted using 10 mM CAPS (3-cyclohexylamino-1-propane sulfonic acid) buffer solution with the pH adjusted to 10.5 using 10 N NaOH. Cry1Fa toxin solution was diluted using 10 mM CAPS buffer solution to a diagnostic concentration of 200 ng cm −2 . The non-treated control was treated with 10 mM CAPS buffer solution only. The diagnostic concentration of Cry1Fa or the non-treated control solution was applied to the diet surface of each well in 30 μl aliquots using a repeater pipette. Trays were tilted in all directions to cover the entire diet surface with solution. Trays were kept in a fume hood as the solvent component of the solution evaporated. A single neonate (< 24 h old) larva was transferred with a fine-paint paint brush to each well of the bioassay tray. Infested wells were covered with adhesive lids, placed into rearing conditions, and covered with cardboard to prevent condensation. A total of 24 larvae were treated per solution per bioassay. Individual larval mortality and weight were recorded after seven days. Stunted larvae that were alive but weighed less than 0.1 mg were considered dead. Each bioassay was repeated five times for each strain.
Introgression experiment. Adults from ON-S1 and NS-R were crossed following the cage method described earlier in the following configurations: 50 ON-S1♀ × 50 NS-R♂ and 50 NS-R♀ × 50 ON-S1♂. Progeny (F 1 ) of the crosses were allowed to mate and their offspring (F 2 ) were exposed to a diagnostic concentration of Cry1Fa as described for the toxin-overlay diet bioassays. Survivors of Cry1Fa exposure were then back-crossed to the ON-S1 strain. This process was repeated five times resulting in a Cry1Fa-resistant strain (BC-R) that is > 96% genetically similar to the ON-S1 strain. Toxin-overlay diet bioassays were conducted with the BC-R strain as described above with 24 and 72 larvae exposed to the zero and diagnostic concentration of Cry1Fa, respectively, and replicated five times. Restriction enzyme genotype analysis was conducted on 25 randomly selected larvae from all five replications from each of the following categories: survived exposure to 0 ng Cry1Fa cm −2 , died after exposure to 200 ng Cry1Fa cm −2 , and survived exposure to 200 ng Cry1Fa cm −2 .
Sequencing. To determine single nucleotide polymorphisms (SNPs) in the ABCC2 gene associated with susceptibility and resistance to the Cry1Fa toxin, RNA samples from two ON-S1 and two NS-R individuals were sequenced. RNA was extracted using TRIzol™ Plus RNA purification kit (Invitrogen™, Waltham, MA) following manufacturer's instructions. Samples were amplified with SuperScript™ IV One-Step RT-PCR System kit (Invit-rogen™, Waltham, MA) on a VeritiPro™ Thermal Cycler (Applied Biosystems™, Waltham, MA) and primers were designed as described below to amplify the target locus. The cycling conditions included reverse transcription at 50 °C for 10 m, followed by initial denaturation at 98 °C for 2 m, then 40 cycles at 98 °C for 10 s, 55 °C for 10 18 .
Open reading frame (ORF) and amino acid sequence of the ABCC2 protein were predicted using the ORF finder software (https:// www. ncbi. nlm. nih. gov/ orffi nder/) and ExPASy Bioinformatics resource portal (http:// web. expasy. org/ trans late/), respectively. The transmembrane topology was predicted using the Phobius web server 35 . BLAST search of the NCBI Conserved Domains Database (http:// www. ncbi. nlm. nih. gov/ cdd) was used to predict the ATP binding cassettes.
Genotyping assay. Based on the sequencing data, a restriction enzyme digest method was developed to determine the presence and linkage of SNPs in resistant individuals. Genomic DNA was extracted from fifth instars of the ON-S1, ON-S2, NS-R, and QC-R strains, as well as from first instars of the ON-S1 × NS-R and BC-R strains using QuickExtract™ DNA Extraction Solution (Lucigen Corporation, Middleton, WI) following the manufacturer's protocol and quantified using Ultraspec 2100 pro spectrometer (Biochrom Ltd, Cambridge, UK). Samples were amplified using GoTaq G2 Green Master Mix (Promega, Madison, WI), with forward and reverse primers (Sigma-Aldrich, St. Louis, MO) (SI Appendix, Table S3) using VeritiPro™ Thermal Cycler (Applied Biosystems™, Waltham, MA). The cycling conditions included initial denaturation at 95 °C for 5 m, then 37 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 40 s, and a final extension at 72 °C for 5 min. The amplicons were digested using the restriction enzymes listed in SI Appendix, Table S3 (New England Biolab, Whitby, ON) for 1.5 h following the manufacturer's protocol. Five of these restriction enzymes cut the resistant allele at the indicated position, while enzyme MspI cut the susceptible allele at SNP1 (SI Appendix, Table S3). Digest products were loaded on 1.5% agarose gel and separated using 100 V for 1 h. The resulting fragment sizes were analysed using a DirectLoad™ 50-bp ladder (Sigma-Aldrich, St. Louis, MO). Protocol describing the PCR and digest methods is available in SI Appendix.
Data analyses. Toxin-overlay diet bioassays. Mortality at the diagnostic concentration of Cry1Fa was analysed using PROC GLIMMIX in SAS v. 9.4 (SAS Institute, Cary, NC) with strain as the fixed effect and replicate as a random effect. Mortality data followed a beta distribution with a logit link and values of 0 and 1 were replaced with 0.0001 and 0.9999, respectively 36 . Tukey's Honestly Significant Difference (HSD) test was used for multiple treatment comparisons and the α level for statistical significance was set at 0.05.
Genotyping assay. A chi-squared test was conducted using PROC FREQ in SAS v. 9.4 (SAS Institute, Cary, NC) to determine if the genotypes for the individuals tested in the back-cross experiment were in Hardy-Weinberg equilibrium.

Data availability
The data generated and/or analyzed in the current study are available from the corresponding author on reasonable request.